The endothelial cell layer (EC) which lines blood vessel walls from the aorta to the capillaries provides the principal barrier to transport of water and solutes between blood and underlying tissue. ECs are continuously exposed to the mechanical shearing force (shear stress) imposed by flowing blood on their surface. We have shown recently that shear stress has an acute effect on transport properties of EC layers in a well defined cell culture model, and this has been confirmed in different vessels of live animals. Shear-dependent EC transport has important implications for the function of normal microvessels, which must deliver material to tissue in proportion to blood flow in the region of demand. In arteries, shear dependent permeability of macromolecules such as low density lipoprotein has been hypothesized to play a key role in the localization of atherosclerotic lesions. Because of its physiological significance, we propose to use a well-established cell culture model in an engineered shearing device to study the phenomena of shear-dependent EC transport. The specific aims of the proposed research are: 1) To determine the effect of steady and oscillatory shear stress on the hydraulic conductivity (Lp) and macromolecular permeability (Pe) of bovine aortic endothelial cell (BAEC) monolayers. A unique apparatus will be used which allows well-defined steady or oscillatory shear stress to be imposed on BAEC monolayers grown to confluence on porous, polycarbonate filters while Lp and Pe are measured simultaneously. 2) To determine the physical transport pathways that are affected by shear stress, using four techniques: (i) en face fluorescence microscopy to detect the presence of endothelial gaps or leaky interendothelial junctions, (ii) immunofluorescence labeling for ZO-1 protein to determine the status of tight junctions, (iii) electron microscopic observation of colloidal gold-labeled tracers to assess vesicular transport versus paracellular transport, and (iv) partial pronase digestion of the EC glycocalyx to probe the contribution of EC surface glycoproteins to transport barrier function and its shear response. 3) To determine the biochemical mechanism(s) mediating the shear-dependent response of EC transport properties. The role of important second messengers systems (cAMP, cGMP, Ca++) coupled to the plasma membrane through G-protein-dependent receptors will be probed with various inhibitors and other modulators to manipulate the shear-dependent response of EC transport. The role of the cytoskeleton will be assessed with specific agents which disrupt the three major components: microfilaments, micro tubules and intermediate filaments.